Despite various mechanisms for alcohol-mediated organ damage, few animal (rodent) models exist to evaluate those concepts. During the last three years, we hypothesized that elevated acetaldehyde and lipid aldehydes such as cytotoxic 4-hydroxynonenal (HNE) and acrolein along with increased oxidative stress may contribute to the development of alcohol-induced tissue damage, since highly reactive and toxic aldehydes, produced during ethanol metabolism, interact with free amino group of cellular proteins and DNA, usually altering their physiological functions of the targets and initiating auto-immune responses and DNA mutations. Accumulation of acetaldehyde and other carbonyl compounds can be achieved through inhibition of the major aldehyde metabolizing enzyme, the mitochondrial aldehyde dehydrogenase 2 (ALDH2), by either chemical inhibitors or genetic mutation (G to A nucleotide substitution) with a subsequent change in Glu487Lys in ALDH2 protein. Individuals with this genetic variation possess reduced ALDH2 activity through dominant inactivation of the enzyme and show aversive reactions and flushing response to alcohol consumption, as observed in many East Asian people. Because of the problems associated with the chemical inhibitors of ALDH2 such as non-selective interactions with other enzymes and proteins and short duration of action, we have taken genetic approaches. We hypothesized that knock-out mice deficient in mouse ALDH2 gene should not possess ALDH2 activity, leading to extremely high levels of acetaldehyde than the background mice upon alcohol exposure. Under proper experimental conditions, the ALDH2 knock-out mice may be more susceptible to tissue damage caused by a high dose of alcohol and another hepatotoxic agent. In order to test these hypotheses and to develop an animal model simulating human conditions, we used gene disruption techniques to specifically delete the mouse ALDH2 gene. We have produced chimera mice (mixed genotypes), which contain our DNA construct specifically designed to delete the mouse ALDH2 gene. After mating between the positive chimer mice and C57/BL mice, we verified that seven male and eleven female heterozygous mice contained the ALDH2- knockout construct. We are in the middle of mating these F1 mice to produce the homozygous knockout mice followed by confirmation with DNA Southern analysis. Parallel to this genetic approach, we also studied the role of ALDH isozymes against HNE-mediated cell damage, because we observed a differential sensitivity of cultured cells toward cytotoxic HNE. Our results showed that some cells are quite resistant while others are relatively sensitive to HNE. Our immunoblot results revealed that the differential sensitivity toward HNE depends on the presence or absence of ALDH isozymes. For instance, the presence of ALDH2 and ALDH1 isozymes makes the cultured cells resistant to HNE-mediated cell death, while certain cells sensitive to HNE did not contain ALDH isozymes. In addition, inhibition of ALDH isozymes with chemical inhibitors of ALDH such as disulfiram significantly increased the rate of HNE-mediated damage in the resistant cells. These results may suggest an important role of ALDH isozymes in cellular defense mechanism against lipid aldehydes, which are being produced after exposure to toxic chemicals and large doses of alcohol. We are determining the levels of other enzymes involved in the metabolism of HNE to evaluate the role of the ALDH isozymes in the HNE-mediated damage.